Catecholamine outflow from mouse and rat brain slice preparations evoked by nicotinic acetylcholine receptor activation and electrical field stimulation

Abstract

Background and purpose: Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine-induced catecholamine outflow in wild type (WT) and nAChR (beta2 and alpha5) knockout mice for a comparison with rat brain slice preparations.

Experimental approach: Brain slices from rat and mouse hippocampus, parieto-occipital neocortex, and corpus striatum were loaded with either [3H]-noradrenaline or [3H]-dopamine. We provoked catecholamine outflow by electrical field stimulation and nicotinic agonists.

Key results: When set in relation to electrical field stimulation, nicotine-evoked catecholamine release was sizeable in the striatum but low in the neocortex of both rats and mice. [3H]-noradrenaline outflow was, on the other hand, substantial in the rat but low in the mouse hippocampus. About 10% (or less) of nicotine-induced catecholamine release persisted in the presence of tetrodotoxin in all our preparations.

Conclusions and implications: Targeted deletion of the beta2 subunit gene essentially abolished the effect of nicotine, indicating that this subunit is an essential constituent of nAChRs that indirectly (via action potentials) induce catecholamine release from hippocampal and striatal slices in mice. The impact of nAChRs in catecholaminergic projection areas differs between species and has thus to be considered when extrapolating results from animal models to human conditions.

Figure 1

[³H]noradrenaline outflow from mouse (a–c) and rat (d–f) hippocampal slices. (a) Fractional rates of outflow in response to electrical field stimulation (circles) or to the application of 100 μM nicotine (triangles). Data are means±s.e.m. (n=3). (b) [³H]noradrenaline outflow in response to indicated stimuli (shown as a percentage of total radioactivity in slices) in WT mice (n=6–27). (c) [³H]noradrenaline outflow in response to indicated stimuli in β₂-KO mice (n=6–9). Compared to WT mice (b), the outflow induced by 20 μM nicotine was reduced by 95.2% in the KO. (d) Fractional rates of [³H]noradrenaline outflow in response to electrical field stimulation (circles) or to the application of 100 μM nicotine (triangles; n=3). (e) [³H]noradrenaline outflow in response to indicated concentrations of nicotine (calculated EC₅₀: 19.6 μM; n=3). (f) [³H]noradrenaline outflow in response to indicated stimuli (n=3–12). nic, nicotine; TTX, 0.5 μM TTX; MCA, 5 μM mecamylamine.

Figure 2

[³H]noradrenaline outflow from mouse (a) and rat (b, c) parieto–occipital neocortical slices. (a) [³H]noradrenaline outflow in response to indicated stimuli (shown as a percentage of total radioactivity). Data are means±s.e.m. (n=3–6). (b) [³H]noradrenaline outflow in response to indicated concentrations of nicotine (EC₅₀: 7.8 μM; n=3). (c) [³H]noradrenaline outflow in response to indicated stimuli (n=5–15). nic, nicotine; TTX, nicotine in the presence of 0.5 μM TTX; MCA, nicotine in the presence of 5 μM mecamylamine.

Figure 3

[³H]dopamine outflow from mouse (a–c) and rat (d–e) corpus striatum slices. (a) [³H]dopamine outflow (shown as a percentage of total radioactivity) in response to 10 μM nicotine and indicated experimental conditions. Each approach was run with its own set of [³H]dopamine outflow evoked by 10 μM nicotine at control conditions. Data are means±s.e.m. (n=3–15). (b) [³H]dopamine outflow in response to electrical field stimulation (n=16) or 10 μM nicotine (n=17) in WT mice. (c) [³H]dopamine outflow in response to electrical field stimulation (n=11) or 10 μM nicotine (n=6) in β₂-KO mice. (d) [³H]dopamine outflow in response to indicated concentrations of nicotine (EC₅₀, 0.84 μM; n=3–6). (e) [³H]dopamine outflow in response to indicated stimuli (n=3–9). nic, nicotine; TTX, 0.5 μM TTX; no Ca²⁺, Ca²⁺ omitted before and during nicotine application; MCA, 1 and 3 μM mecamylamine; MII, 50 and 100 nM α-CtxMII.

Figure 4

[³H]dopamine outflow in response to nicotine (a), DMPP (b) and cytisine (c) from striatal slices taken from WT (circles, solid lines) or α₅-KO mice (triangles, dashed lines). Data points are means±s.e.m. (n=3–15). Curves were fitted to data points as described in Methods. See Table 2 for EC₅₀ and R_max values.